Project Summary/Abstract The human SWItch/Sucrose NonFermentable chromatin-remodeling complex, commonly abbreviated as SWI/SNF, is composed of 10-15 biochemically distinct subunits. SWI/SNF complexes use the energy provided from ATP to reposition nucleosomes and modulate transcription. We found that SWI/SNF chromatin- remodeling genes are mutated in 20% of all cancers, including 30% of melanomas. While genetic studies clearly implicate SWI/SNF genes as tumor suppressors, it is not clear how mutations in these genes contribute to cancer. Experimental evidence from our group and others indicates that the genetic context in which a SWI/SNF component is perturbed heavily influences the fate of that cell. It will be important to study these mutations in their proper genetic context in order to resolve their functional contributions to cancer. Unfortunately, the genetic context in which SWI/SNF mutations occur is poorly understood. This is because tumors evolve through a multistep process, and most tumors are sequenced at a late stage, after they have fully evolved. I have developed an assay to determine the order of mutations as they occur during the evolution of melanoma. In aim 1 will utilize this assay to determine the precise context in which SWI/SNF mutations occur during the evolution of melanoma. This is innovative because I utilize a cutting-edge approach to reveal an unresolved feature of SWI/SNF tumor biology. In aim 2, I will engineer primary human melanocytes using CRISPR/Cas9 to mimic the genetic context prior to and after a SWI/SNF mutation occurs. These parental and SWI/SNF-mutant daughter cell lines will then be compared to functionally and mechanistically interrogate the effects of SWI/SNF mutations in an otherwise isogenetic background. This strategy is innovative because there are no known cell lines that definitively recapitulate the genetic context of a partially evolved melanoma before and after a SWI/SNF mutation occurs. I hypothesize that SWI/SNF mutations occur in a reproducible context during melanoma progression and promote either an invasive or a mutator phenotype. This hypothesis is based on observations from preliminary data that SWI/SNF mutations occur at the transition to invasive melanoma and coincide with a spike in the overall mutation burden. This proposal is significant because SWI/SNF genes are frequently mutated in melanoma, yet almost nothing is known regarding their roles in melanomagenesis. SWI/SNF proteins are not themselves ideal drug targets because they are tumor suppressors and are also important in normal cellular biology; however, completion of these studies should reveal the mechanisms underlying SWI/SNF melanoma biology and thus provide therapeutic opportunities for the large number of patients whose tumors are driven by these mutations. Finally, the protected time associated with this unique award mechanism will allow me to carry out scientific and professional career development activities, as described, that will facilitate the success of my independent research career.